The overall objective of this study is to identify specific and potent modulators of the two-pore-domain potassium channel, TREK-2. TREK-2 channels are important regulators of cellular electrical excitability and serve diverse physiological roles. TREK-2 is expressed predominately in the nervous system and pancreas, with strong expression in DRG neurons and neuroendocrine cells. TREK-2 channel activity may, thus, regulate stimuli detection of sensory neurons and neuroendocrine hormone secretion. However, our understanding of the role(s) of TREK-2 in human tissues remains obscure due to a lack of specific and potent pharmacology. Therefore, this project will utilize a robust thallium (Tl+) based fluorescent assay in a high throughput screen (HTS) to identify small molecule modulators of the human TREK-2 channel. The assay will be performed on a tetracycline inducible TREK-2 cell line, which was selected for based on its performance in the Tl+ assay. The TREK-2 Tl+ assay was validated with primary screens of two small molecule libraries including the Spectrum Collection (~2000 molecules) and a bioactive lipid library (~1000 molecules), which identified a small cohort of molecular regulators of TREK-2. The primary screens were utilized to optimize the Tl+ assay for use with the TREK-2 cell line in a large HTS. Building on these preliminary studies, this proposal plans to perform a HTS on the human TREK-2 channel with the diverse small molecule library at the Johns Hopkins Ion Channel Center within the Molecular Libraries Probe Centers Network. This will be accomplished using 1. A Tl+ flux based HTS, which will be followed by 2. Secondary assays utilizing Tl+ flux as well as electrophysiology to support rapid hit-to-lead progression and finally 3. A battery of tests including biophysical analysis, mutagenesis and phosphorylation analysis together with Tl+ flux; these will determine the mechanism of action, specificity and potency of the small molecule regulators of TREK-2. Molecular regulators of TREK-2 identified in this HTS will be utilized to test the influence of TREK-2 channels on human islet cell electrical activity and hormone secretion.